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Configuration

Learn how to setup a configuration file required bycelseq2 as input. The configuration file is global and can be reusable.

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Why configuration

Global configuration file has 3 purposes:

1. Tell celseq2 how CEL-Seq2 is performed. For example, what are the sequences of the cell barcodes in use?
2. Specify bioinformatics details of celseq2. For example, what is the absolute path to the genome annotation on your computer?
3. Configuration file is reusable as long as both experimental and bioinformatics protocol are applied on same type of species.

Create a configuration file from template

celseq2 provides a bash command new-configuration-file to initiate a new configuration template so that user can fill in details.

new-configuration-file -o /path/to/my_wonderful_config.yaml

How to specify configuration

Here is a real example as global configuration.

###########################
## CEL-seq2 Tech Setting
###########################
BC_INDEX_FPATH: 'yanailab/refs/barcodes/barcodes_cel-seq_umis96.tab'
BC_IDs_DEFAULT: '1-96'
UMI_LENGTH: 6
BC_LENGTH: 6

###########################
## Bowties Index
###########################
BOWTIE2_INDEX_PREFIX: 'yanailab/refs/danio_rerio/danRer10_87/genome/Danio_rerio.GRCz10.dna.toplevel'
BOWTIE2: '/local/apps/bowtie2/2.3.1/bowtie2'

###########################
## Annotations
###########################
GFF: 'yanailab/refs/danio_rerio/danRer10_87/gtf/Danio_rerio.GRCz10.87.gtf.gz'

###########################
## Demultiplexing
###########################
FASTQ_QUAL_MIN_OF_BC: 10
CUT_LENGTH: 35

###########################
## Alignment
###########################
ALIGNER: 'bowtie2'

###########################
## UMI Count
###########################
ALN_QUAL_MIN: 0

Explanations of key parameters

BC_INDEX_FPATH

Absolute file path to a space/tab separated file which saves all the sequences for cell barcodes.

Here are first 11 lines of the content of BC_INDEX_FPATH

#barcode_id sequence
1   AGACTC
2   AGCTAG
3   AGCTCA
4   AGCTTC
5   CATGAG
6   CATGCA
7   CATGTC
8   CACTAG
9   CAGATC
10  TCACAG

UMI_LENGTH, BC_LENGTH, CUT_LENGTH

CEL-Seq2 sequences in a pair-end manner. Read-1 records the sequences of UMIs and cell barcodes, while read-2 records the sequences of RNA transcripts. celseq2 will cut a subsequence with length of CUT_LENGTH since the left-most end of read-2, which will be ready for alignment.